1. Field of the Invention
The present invention relates to an improved stage assembly for a microscope. The stage assembly includes a perfusion fluid preheater/cooler assembly and a coverslip chamber assembly mounted on a mechanical stage for a microscope.
More specifically, the present invention relates to an improved heating/cooling or warming stage including a perfusion fluid pre-heater/cooler assembly and an improved specimen chamber/coverslip assembly.
2. Description of the related art including information disclosed under 37 CFR 2121 1.97-1.99
Various techniques, methods and apparatus have been proposed for controlling the temperature of a specimen on a microscope stage.
The present invention relates to a tissue culture vessel for high power examination of cells and tissues on a microscope.
Current problems associated with apparatus now in the marketplace relate to their large chamber circumferences and their large respective volumes. A primary goal of previous designs has been to allow the use of high magnification and oil immersion objective lenses having extremely short working distances on inverted microscopes. To achieve this, the underside circumference of the chamber vessel must allow for sufficient scanning movement of the objective lens at very close proximity to the coverslip. (Working distances for oil immersion objective lenses can be as little as 0.14 mm/0.0055"). Unfortunately, these designs resulted in chamber perimeters substantially identical to the sub-chamber perimeters. (See U.S. Pat. No. 4,435,508 described below--the Chamber/Dish by Bionque Laboratories--The Microscope Tissue Chamber by Biophysica Technologies Inc.--and the Leiden Coverslip Dish sold through Medical Systems Corp.) Typical chamber diameters are at least 25 mms for these products.
One undesirable situation occurs when a rapid solution exchange is required, especially when the perfused fluid contains an expensive, experimental drug. A large volume slows the solution exchange rate increasing downtime between achieving the desired drug concentration and the intracellular response. When this procedure is repeated several times to verify a causal response, a significant amount of the experimental drug is consumed.
A second drawback relates to the thermal inertia of the available tissue chamber apparatus. The chamber wall surfaces are constructed of a combination of chemically inert substances, such as Teflon and silicone rubber in substantially thick sections. Both materials, having low coefficients of thermal conductivity, slow the transfer of thermal energy to the chamber bath.
A third problem relates to temperature gradients common in radially heated or cooled static chamber baths. The larger the chamber circumference, the larger the temperature gradient.
Some of the devices which must access the chamber bath, such as perfusion tubing, aspiration tubing, and a temperature probe, have conventionally been positioned using magnetic-based holders or with a magnetic ring surrounding the chamber. These have typically been bulky devices which compete for available space with micromanipulator devices, microelectrodes, etc.
During perfusion procedures, such as when continuous nutrient/waste exchanges occur and when therapeutic agents are introduced intermittently, a need arises for the thermoregulation of the perfused medium prior to its entry into the chamber bath. Without the prior heating/cooling of the perfused medium, both temperature oscillations and gradients will occur in the chamber bath. Conventional methods heat or cool the medium to a preset temperature at a point removed from the chamber bath. Through various means, such as pumping, or a controlled gravity flow, the medium is transported to the chamber bath via various types of tubing. If the transportation distance is anything but very short, the tubing itself will act as a thermal sink and affect the perfusion medium temperature.
Another associated problem occurs during an experiment requiring a quick and simultaneous raising or lowering of the chamber bath and perfusion medium temperatures. Even with the use of two independent, time and temperature programmable controller systems, uniform temperature tracking is difficult.
One proposed apparatus for maintaining thermal control of the temperature of a microscope stage is disclosed in the Middlebrook U.S. Pat. No. 4,888,463. This patent discloses a heating device which comprises a planar member having a pattern of non-inductive electrical heating means on one surface thereof adapted to be placed onto the stage of a microscope for providing heat to a specimen within a container mounted on the stage.
Also, there is disclosed in the Kitagawa et al U.S. Pat. No. 4,629,862 a sample heater for use in microscopes which includes a platform supporting thereon a sample container and a heater for heating at least a part of the platform.
Further, there is disclosed in the Papas U.S. Pat. No. 4,195,131 for use with a tissue or specimen receiving vessel such as a petri dish, an environmentally controlled unit which includes a sealable chamber in blocks of the unit which have passageways through and around the sealable chamber for providing a controlled flow of fluid throughout the chamber and an additional flow of temperature controlling fluid within the block of the unit adjacent to the blocks containing the sealable chamber.
Still further, there is disclosed in the Gabridge U.S. Pat. No. 4,435,508 a tissue culture vessel comprising two rigid plates with aligned apertures with the upper plate separated from the lower plate by a rubber gasket.
As will be described in greater detail hereinafter, the heating/cooling or warming stage with specimen chamber coverslip assembly and perfusion fluid preheater/cooler assembly of the present invention differs from the previously proposed microscope stages with heating systems therefor by providing, on a stage support plate, a perfusion fluid preheating/cooling assembly for maintaining the temperature of the perfusion fluid injected into the specimen chamber above the coverslip at the same temperature as the temperature of the fluid in the specimen chamber and by providing an improved plate containing the specimen chamber mounted above the coverslip on the stage support plate.
The heating/cooling or warming stage with coverslip assembly of the present invention provides a coverslip chamber assembly which includes a specimen chamber which is mounted on a stage support plate mounted on a mechanical stage and thermally coupled to the stage support plate which is electrically and thermally isolated from the mechanical stage.
Also, the heating/cooling or warming stage with coverslip assembly of the present invention provides a specimen chamber of reduced diameter and volume capacity, while maintaining the standard sized subchamber diameter necessary for optical scanning when using high magnification or oil immersion objective lenses.